The rise in obesity related diseases such as Non-Alcoholic Fatty Liver Disease (NAFLD) and Hepatocellular Carcinoma (HCC), which has a 4.5 fold increased risk of death in U.S. obese patients, highlights our deficit in knowledge of signaling cascades that link metabolic stress to the basic transcriptional machinery. Additionally, the molecular pathways that influence the transition of NAFLD to HCC are poorly defined. The rationale for this project is that a comprehensive understanding of the normal transcriptional mechanisms that respond to metabolic stress would provide valuable insight into how metabolic stress impacts the transcriptional landscape in the progression from NAFLD to HCC. To better define the molecular pathways that contribute to the development of obesity-related diseases, we used Steroid Receptor Coactivator 2 (SRC-2), a member of the p160 family of steroid receptor coactivators, to investigate normal and diseased transcriptional mechanisms. The literature supports that SRC-2 has an essential role in regulating hepatic glucose and lipid metabolism, both of which are perturbed in the pathobiology of NAFLD. Even though little is known about the upstream signaling inputs to SRC-2, AMP-activated protein kinase alpha 2 (AMPK?2) influences the intrinsic transcriptional activity of SRC-2. Additionally, loss of either hepatic SRC-2 or AMPK?2 results in liver lipid accumulation. Taken together, these findings suggest that AMPK?2 and SRC-2 may functionally cooperate to coactivate critical gene programs that control nutrient-dependent transcriptional responses. Therefore, we hypothesize that SRC-2 and AMPK?2 interact on promoters of key metabolic genes to impact recruitment and activity of other transcriptional proteins, and this interaction is disrupted during the pathogenesis of NAFLD. To investigate the mechanism by which SRC-2 and AMPK?2 coregulate metabolic gene expression, we have designed a DNA pull-down strategy wherein whole liver nuclear extracts are incubated with a biotin labeled target promoter followed by mass spectrometry (MS) and immunoblot validation to identify regulatory components of the SRC- 2-AMPK?2 promoter complex. Building on our preliminary data, we are confident the proposed approach will permit functional characterization of the transcriptional complexes specified by SRC-2 and AMPK?2 that dictate metabolic gene expression. Moreover, the proposed strategy will unveil previously unappreciated signaling components that function to link metabolic stress to transcriptional output via the actions of SRC-2 and AMPK?2. We will also determine the involvement of the SRC-2 and AMPK?2 interaction during the pathogenesis of NAFLD to HCC using banked human liver biopsies of patients obtained during progression of disease in the same patient from NAFLD to HCC. Definition of the normal SRC-2 and AMPK?2 interactions as well as the molecular and phenotypic outcome of metabolic stress on the axis may eventually lead to a targeted NAFLD therapeutic.